1-(3-Methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one was another derivatives compound from 1,6-dihydroxyxanthone. This compound was synthesized through the reaction between 1,6-dihydroxyxanthone and prenyl bromide in the presence of potassium carbonate in aqueous medium. Potassium carbonate acted as both a base and a strong catalyst to lower the free energy of activation so that the forward rate of reaction can be increased. The total mass of pure 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one isolated was 0.0800 g, which was 6.1% of the percentage yield.
Other than 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9- one, another major compound isolated from this synthesis was 1-hydroxy-6-(3- methyl-but-2-enyloxy)-xanthen-9-one which was identical to the compound produced in the former prenylation of 1,6-dihydroxyxanthone in organic acetone medium. The total mass of pure 1-hydroxy-6-(3-methyl-but-2-enyloxy)-xanthen- 9-one obtained from the aqueous-medium based synthesis was 0.1847 g, or 17.3%
of the percentage yield. When TLC plate was developed with solvent system of 85% hexane and 15% acetone, the single red spot gave a Rf value of 0.30. The overall equation of reaction is shown in Figure 4.16.
74
O
O
HO
OH
Br
O
O OH
O O
O
O
O
Figure 4.16: Equation of reaction for prenylation in aqueous medium
4.4.1 Structure Elucidation of 1-(3-Methyl-but-2-enyloxy)-6-(3-methyl-but-2- enyloxy)-xanthen-9-one
1-(3-Methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one was isolated in the form of yellowish amorphous with a melting point range of 68oC to 70oC. When the TLC was developed with solvent system of 85% hexane and 15%
acetone, the collected fractions from 4 to 7 gave a single blue spot with Rf value of 0.39. The summary of physical properties of both compounds 1-(3-methyl-but- 2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one and 1-hydroxy-6-(3- methyl-but-2-enyloxy)-xanthen-9-one is tabulated in Table 4.6.
+
+
1-hydroxy-6-(3-methyl-but-2- enyloxy)-xanthen-9-one
1-(3-methyl-but-2-enyloxy)-6-(3-methyl- but-2-enyloxy)-xanthen-9-one
K2CO3
H2O
75
Table 4.6: Summary of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2- enyloxy)-xanthen-9-one and 1-hydroxy-6-(3-methyl-but-2- enyloxy)-xanthen-9-one
O O
O
O
O
O OH
O
Mass obtained, g 0.0800 0.1847
Percentage yield, %
6.1 17.3
Physical appearance
Yellowish amorphous Yellowish solid
Melting point 68oC to 70oC 123oC to 125oC Rf value on TLC 0.39 in hexane:acetone (85:15) 0.30 in hexane:acetone
(85:15)
The 1H-NMR analyses (Figure 4.17) of pure 1-(3-methyl-but-2-enyloxy)-6-(3- methyl-but-2-enyloxy)-xanthen-9-one revealed that two prenyl groups were substituted to the compound. The presence of two pairs of characteristic signals 4.47(2H,d,J=6.7 Hz) and 5.35(1H,t,J=6.7 Hz), 4.57(2H,d,J=6.7 Hz) and 5.48(1H,t,J=6.7 Hz) in the range from 4.0 ppm to 6.0 ppm signified the presence of two O-prenyl groups in structure of compound. The absence of signal in the range from δ 12.0 to 13.0 suggested the chelated hydroxyl group bonded to
76
carbon C-1 was reacted during the synthesis. This further confirmed that one of the prenyl groups was attached at the O-position of carbon C-1. The presence of six resonances at δ 8.03 (1H, d, J=9.2 Hz), 7.40 (1H, t, J=8.2 Hz), 6.86 (1H, d, J=8.2 Hz), 6.74 (1H, d, J=9.2 Hz), 6.67 (1H, d, J=2.4 Hz), and 6.65 (1H, d, J=8.2 Hz) were assigned to protons H-8, H-3, H-4, H-7, H-5, and H-2, respectively. The olefinic protons H-17 and H-12 in the prenyl groups gave two triplet signals at 5.36 ppm (1H, t, J=6.7Hz) and 5.48 ppm (1H, t, J=6.7 Hz), respectively. Two doublet signals at 4.58 ppm (2H, d, J=6.7 Hz) and 4.47 ppm (2H, d, J=6.7 Hz) were assigned to the benzylic methylene protons H-11 and H-16, respectively.
The two intense singlet signals at 1.67 ppm and 1.63 ppm each integrated for six protons were respectively assigned to the methyl proton H-14&-15 and H-19&-20 in the two prenyl groups. The aromatic proton H-8 had a relatively higher chemical shift compared with others due to the deshielding effect by its neighbouring carbonyl group.
From 13C-NMR spectrum (Figure 4.18) of this pure compound, a peak at 175.6 ppm was assigned to carbon C-9 which was highly deshielded carbonyl carbon.
The presence of nine carbon signals at 175.6 ppm, 163.8 ppm, 159.9 ppm, 158.2 ppm, 156.7 ppm, 139.1 ppm, 137.4 ppm, 116.8ppm, and 112.7 ppm were assigned to quaternary carbon C-9, C-6, C-1, C-4a, C-10a, C-18, C-13, C-8a, and C-9a, respectively. Besides, another eight carbon signals at 134.2 ppm, 128.1 ppm, 119.6 ppm, 118.7 ppm, 113.3 ppm, 109.6 ppm, 107.0 ppm, and 100.2 ppm were assigned to methine carbons C-3, C-8, C-12, C-17, C-7, C-4, C-2, and C-5,
77
respectively. Two methylene carbons in both prenyl group gave two doublet signals at 66.4 ppm and 65.3 ppm which were assigned to carbon C-11 and C-16, respectively while the carbon signals at 25.7 ppm and 18.3 ppm were assigned to methyl carbons C-14&-15 and C-19&-20.
The assignment of chemical shifts in the 1H-NMR and 13C-NMR of 1-(3-methyl- but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one was further confirmed by 2D-NMR spectroscopy such as HMQC and HMBC analyses.
From HMQC spectrum (Figure 4.19), the six aromatic methine carbon signals at 134.2 ppm, 128.1 ppm, 113.3 ppm, 109.6 ppm, 107.0 ppm and 100.2 ppm were correlated to the proton signals at 7.40 ppm, 8.03 ppm, 6.74 ppm, 6.86 ppm, 6.65 ppm, and 6.67 ppm, respectively. Besides, two methylene carbons at both prenyl group having signals at 66.4 ppm and 65.3 ppm were found to show connectivity to proton signals at 4.58 ppm and 4.47 ppm, respectively. The methine prenyl carbons at 119.6 ppm and 118.7 ppm were attached to proton signals at 5.48 ppm and 5.36 ppm, respectively. The two methyl carbons in both the prenyl side chain having signals at 25.7 ppm and 18.2 ppm was respectively connected to proton resonaces at 1.67 ppm and 1.63 ppm, respectively.
78
In this HMBC spectrum (Figure 4.20), the benzylic methylene proton H-11 (4.58 ppm) showed long-range heteronuclear connnectivities with an oxygenated aromatic carbon C-1 (159.9 ppm), quaternary carbon C-13 (137.4 ppm), and prenyl methine carbon C-12 (119.6 ppm) indicating the first prenyl group was attached to the xanthone skeleton through the oxygen atom bonded to carbon C-1.
On the other hand, the benzylic methylene proton H-16 (4.47 ppm) showed 3J coupling with oxygenated aromatic carbon C-6 (163.8 ppm), 3J coupling with quaternary prenyl carbon C-18 (139.1 ppm), and 2J coupling with methine prenyl carbon C-17 (118.7 ppm). The aromatic methine carbon C-8 showed correlation with oxygenated carbon C-6 (163.8 ppm), carbonyl carbon C-9 (175.6 ppm), and quaternary carbon C-10a (156.7 ppm) with 3J couplings. These correlations again confirmed the presence of two units of prenyl side chain which were attached to O-position at carbons C1 and C6.
The IR spectrum (Figure 4.21) of the compound showed the absence of broad peak in the region 3600 - 3200 cm-1, this implied that there was absence of –OH functional group in the molecule. In fact, this was expected in 1-(3-methyl-but-2- enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one as the proton in both the hydroxyl groups were substituted with prenyl groups. The intense peak at 1618 cm-1 was indication of the presence of C=O functional group in the compound.
79
Using UV-Vis spectrophotometry analysis, the isolated compound gave absorption maxima at 242.93 nm, 289.88 nm, and 339.39 nm (Figure 4.22), indicating that the isolated compound was highly conjugated and is agreement with the proposed structure 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2- enyloxy)-xanthen-9-one.
80
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Table 4.7: Summary of NMR spectral data of 1-(3-methyl-but-2-enyloxy)-6- (3-methyl-but-2-enyloxy)-xanthen-9-one
Position 1H δ (ppm) 13C δ (ppm) HMBC
1 - 159.9
2 6.65(1H,d,J=8.2 Hz) 107.0 C-4(3J),9a(3J)
3 7.40(1H,t,J=8.2 Hz) 134.2 C-1(3J)
4 6.86(1H,d,J=8.2 Hz) 109.6 C-4a(2J), 2(3J)
4a - 158.2
5 6.67(1H,d, J=2.4 Hz) 100.2 C-6(2J),10a(2J),8a(3J)
6 - 163.8
7 6.74(1H,d,J=9.2 Hz) 113.3 C-8a(3J), 5(3J) 8 8.03(1H,d,J=9.2 Hz) 128.1 C-9(3J),6(3J),10a(3J)
8a - 116.8
9 - 175.6
81
9a - 112.7
10a - 156.7 -
11 4.58(2H,d,J=6.7 Hz) 66.4 C-1(3J),13(3J),12(2J) 12 5.48(1H,t,J=6.7 Hz) 119.6
13 - 137.4
14 1.63(3H,s) 18.3 C-12(3J),13(2J),15(3J)
15 1.64(3H,s) 25.7 C-12(3J),14(3J)
16 4.47(2H,d,J=6.7 Hz) 65.3 C-6(3J),18(3J),17(2J) 17 5.36(1H,t,J=6.7 Hz) 118.7
18 139.1 -
19 1.63(3H,s) 18.3 C-17(3J),18(2J),20(3J)
20 1.67(3H,s) 25.7 C-17(3J),19(3J),
82
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.17: 1H-NMR spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl- but-2-enyloxy)-xanthen-9-one (400 MHz, CDCl3)
H14 & H19 H15& H20
H16 H11 1 H17
H12 H5 H2 H3 H4 H8
H7
83
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.18: 13C-NMR spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl- but-2-enyloxy)-xanthen-9-one (100 MHz, CDCl3)
C14 & C19 C15 & C20 C16
C11 C5 1
C7 C4 C9a C17
C12 C3 C8 C13
C18 C10a C1 C9 C6
C4a
C2
84
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.19: HMQC spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl- but-2-enyloxy)-xanthen-9-one
H8 H11 H16 H14&H19
85
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.20: HMBC spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but- 2-enyloxy)-xanthen-9-one
H14&H19 H11 H16
H8
86
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.21: IR spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2- enyloxy)-xanthen-9-one
4000.0 3000 2000 1500 1000 400.0
16.9 20 25 30 35 40 45 50 53.6
cm-1
%T
29632922 2855
23652345
1655 1637
1618 16011458
1437 13761367
1333
12671223 1179
11031091
1064 1015978
944 902838
827 795
767 717690
670610 485
443
C=O stretch
87
O O
O
O 1
2
3 4 10a 4a
9 9a 8a
5 6 7
8
11 12
13 14
16
15
17 19
20 18
1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one Molecular formula: C23H24O4
Molecular weight: 364.192 g mol-1
Figure 4.22: UV-Vis spectrum of 1-(3-methyl-but-2-enyloxy)-6-(3-methyl- but-2-enyloxy)-xanthen-9-one
190.0 250 300 350 400.0
0.03 0.5 1.0 1.5 2.0 2.5 3.0 3.5 3.74
nm A
JYP-3
339.39,2.4870 289.88,2.9962
242.93,3.7364
88
4.4.2 Proposed Mechanism for Synthesis of 1-(3-Methyl-but-2-enyloxy)-6-(3- methyl-but-2-enyloxy)-xanthen-9-one
The outline of proposed mechanism to account for the formation of 1-(3-methyl- but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one is as follow:
Dissociation of potassium carbonate in aqueous:
K2CO3 2 K+ + CO32-
O O
O
O H
CO32-
H
CO32-
Br
O O
O
O: :
.. -
..
..
: -
Br
O O
O
O
Figure 4.23: Reaction mechanism involved in the synthesis of 1-(3-methyl- but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one
89 4.5 Bioassay
The xanthone block, 1,6-dihydroxyxanthone and its derivatives, 1-hydroxy-6-(3- methyl-but-2-enyloxy)-xanthen-9-one and 1-(3-methyl-but-2-enyloxy)-6-(3- methyl-but-2-enyloxy)-xanthen-9-one were synthesized and tested for their cytotoxic activities toward HeLa and MDA-MB-231 cancer cell lines. The cytotoxicity of xanthone block and its derivatives were evaluated according to the cell viability percentage of HeLa and MDA-MB-231 cancer cells at various concentrations by using MTT method. Structure-activity relationships can be established from the analysis of the IC50 values of xanthone block and its derivatives. The value of half maximal inhibitory concentration, IC50 for each compound was obtained through the graph of cell viability against concentration of drug and these values were summarized in Table 4.8.
Cell viability is a determination of either living or dead cell, based on a total cell sample. In this research, cell viability measurement by MTT method was used to determine the effectiveness of a particular drug due to its toxicity towards the cancer cell line. In fact, cell viability is low when an extremely effective drug is treated with it, which means the cell survival is very low. The half maximal inhibitory concentration, IC50 is a measurement of the effectiveness of a drug in inhibiting biological function of a cell. This quantitative measurement gives the concentration of a particular drug required to inhibit the biological process of
90
given cancer cells by half. Low value of IC50 indicates the drug is very effective as low concentration of drug is sufficient to inhibit cell growth.
Table 4.8: Cytotoxicity of xanthone and its derivatives against HeLa and MDA-MB-231 cancer cell lines
Inhibitory concentration, IC50 (μg / ml) HeLa cancer cell line MDA-MB-231 cancer
cell line
1,6-dihydroxyxanthone 7.0 > 50
1-hydroxy-6-(3-methyl-but- 2-enyloxy)-xanthen-9-one
> 50 > 50
1-(3-methyl-but-2-enyloxy)- 6-(3-methyl-but-2-enyloxy)- xanthen-9-one
> 50 >50
The first observation from Table 4.8 is that the HeLa cancer cell line was found to be highly susceptible towards 1,6-dihydroxyxanthone with IC50 value of 7.0 μg / ml. Meanwhile, both the derivatives of xanthone, 1-hydroxy-6-(3-methyl-but-2- enyloxy)-xanthen-9-one and 1-(3-methyl-but-2-enyloxy)-6-(3-methyl-but-2- enyloxy)-xanthen-9-one gave no significant inhibitory activity towards HeLa cancer cell line. This result revealed that the cytotoxic activity was depend on the presence of hydroxyl group in the xanthone. Inhibitory activity was also reported to show significant dependence on the number of hydroxyl group (Liu et al.,
Compound
91
2006). For 1-hydroxy-6-(3-methyl-but-2-enyloxy)-xanthen-9-one and 1-(3- methyl-but-2-enyloxy)-6-(3-methyl-but-2-enyloxy)-xanthen-9-one, the hydrogen atom of hydroxyl group on the ring was replaced by the prenyl group during etherification which resulted in a significant decrease in cytotoxic activity. In addition, Liu et al also stated that, in order to achieve significant inhibitory activities, there must be three or more hydroxyl group attached to the xanthone ring. This was well consistent with the result of this project where the prenylated xanthones with less hydroxyl substituents were shown to have greatly reduced cytotoxic activity.
Besides the screening on HeLa cancer cells, the three compounds were also subjected to cytotoxic assay against MDA-MB-231 cancer cell line. However, all the tested compounds gave insignificant inhibitory activity against the cell line with IC50 value > 50 μg/mL. This result suggested that the disubstitued prenyl group on the oxygen atom of the xanthone backbone was not essential for eliciting inhibitory activities toward MDA-MB-231 cell lines. Therefore, further researches need to be carry out in order to have better understanding on how 1,6- dihydroxyxanthone exerts its cytotoxic activity on Hela cancer cell line.
92
Figure 4.24: Graph of cell viability against concentration of 1,6- dihydroxyxanthone (HeLa cancer cell line)
0 20 40 60 80 100 120
0 10 20 30 40 50 60
Cell Viability, %
Concentration of drug, μg/ml